Apoptosis is a physiological mechanism of cell death which involves the fragmentation of a cell into membrane-bound particles. The process of apoptosis is involved in a variety of normal and pathogenic biological events, both during development and in adulthood. Agents which affect apoptosis may have therapeutic utility in treating diseases and disorders characterized by aberrant cell proliferation or death (reviewed in Hoeppner et al., Biochim. Biophys. Acta 1242: 217-220, 1966; Thompson, Science 267:1456-1462, 1995). Techniques for detection of apoptosis may be useful to screen for potential therapeutic agents that may induce or prevent apoptosis.
In view of the biological importance of apoptosis, there exists a need for methods to specifically detect cells undergoing apoptosis and those which have suffered apoptotic cell death. These methods are crucial to the identification, characterization, and diagnosis of diseases distinguished by abnormal apoptosis, and to the screening of potential therapeutic agents that may induce or prevent apoptosis.
Several methods are known for the detection of apoptosis in vitro and in vivo, but these have significant drawbacks which limit their utility. Commonly, apoptosis is characterized by condensation and margination of nuclear chromatin, and fragmentation of nuclear structure into so-called apoptotic bodies. This apoptotic morphology can be observed using conventional stains, dyes which selectively accumulate in nuclei such as propidium iodide or Hoechst 33258, or by electron microscopy (e.g., Nicoletti et al., J. Immunol. Methods 139:271-279 1991; Crompton et al., Biochem. Biophys. Res. Commun. 183:532-537 1992; Frey, Cytometry 21:265-274 1995; Woo, N. Engl. J. Med. 333:18-25 1995). Unfortunately, these techniques are either of insufficient specificity or are too laborious and technically complex for the routine selective identification and quantification of apoptotic cells in situ.
Recent attempts to identify and quantify apoptosis have taken advantage of the internucleosomal fragmentation of DNA which is often linked to, but is not diagnostic for, cell death by apoptosis. Various in situ histochemical techniques have been applied to the end-labeling of nicked DNA (Gavrieli et al., J. Cell Biol. 119:493-501 1992; Wijsman et al., J. Histochem. Cytochem. 41:7-12 1993; Wood et al., Neuron 11:621-632 1993). Although these techniques have become popular for marking apoptotic cells in situ, it has become recognized recently that DNA fragmentation can also result from cell stress or necrotic degeneration. Consequently, the in situ techniques which detect fragmented DNA are not selective in detecting cells undergoing apoptosis (Nitatori et al., J, Neurosci. 15:1001-1011 1995; Lassmann et al., Acta Neuropathol, 89:35-41 1995).
Molecular techniques have also been employed for the detection in cell and tissue extracts of internucleosomal DNA degradation linked to apoptosis (Wyllie, A H, Nature 284:555-556 (1980); Wyllie et al., J. Pathol. 142:67-77 (1984)). The in situ and molecular techniques which rely on the detection of internucleosomal DNA fragmentation are not sufficiently thorough for the detection of apoptotic cell death since they do not detect forms of apoptosis not associated with internucleosomal DNA degradation (Cohen et al., Biochem. J. 286:331-334 1992; Schulze-Osthoff et al., J. Cell Biol. 127:15-20 1994). Moreover, the molecular methods lack the sensitivity and cellular resolution needed to define the role of apoptosis of particular cell types in disease processes. This is especially true for chronic slow degenerative diseases, in which cell death is protracted and asynchronous, and individual apoptotic cells are present for only a limited period of time.
An increased understanding of the biochemical mechanisms of apoptotic cell death has arisen from recent genetic and cell biological studies. A family of cysteine proteases related to interleukin-1β converting enzyme (ICE) has been found to play an essential role in the intracellular pathway of apoptosis (reviewed in Martin et al., Cell 82:349-352 1995). ICE itself is not a mediator of apoptosis in most mammalian cell types. Rather, a family of homologous proteases comprising at least nine human ICE family proteases have been identified to date (ICE, CPP32/apopain/Yama, ICH-1, TX/ICH-2/ICErelIII, ICErelIII, MH-1/MH-3/ICE-LAP3, Mch2, FLICE/Mch5, ICE-LAP6/Mch6), each of which leads to apoptosis when over-expressed in a proteolytically active form in cultured mammalian cells (Miura et al., Cell 75:653-660 1993; Wang et al., Cell 78:739-750 1994; Fernandes-Alnemri et al., J. Biol. Chem. 269:30761-30764 1994-Faucheu et al., EMBO J. 14:1914-22 (1995); Kamens et al., J. Biol. Chem, 270:15250-15256 1995, Alnenui et al., J. Biol. Chem. 270:4312-4317 1995; Fernandes-Alnemri et al., Cancer Res. 55:6045-6052 1995; Lippke et al., J. Biol. Chem. 271:1825-1828 1996; Muzio et al., Cell 85:817-827 1996; Duan et al., J. Biol. Chem. 271:16720-16724 1996). Moreover, treatment of cells with apoptotic stimuli increases ICE-like proteolytic activity in cell extracts (Los et al., Nature 375:81-83 1995; Enari et al., Nature 380:723-726 1996). Proteolytic activity by ICE homologues is required to initiate apoptosis, since overexpression of mutant, inactive ICE homologues does not lead to apoptosis, and several protease inhibitors of the ICE family block apoptosis (Miura et al., ibid.; Gagliardini et al., Science 263:826-828 1994; Enari et al., Nature 375:78-81 1995; Milligan et al., Neuron 15:385-393 1995, Los et al., ibid.; Zhivotovsky et al., Exp. Cell Res. 221:404-412 1995; Schlegel et al., J. Biol. Chem. 271:1841-1844 1996).
Degradation of specific cellular proteins, as would be expected to occur following the activation of an ICE-like protease, has also been associated with apoptosis. For example, poly(ADP-ribose)polymerase (PARP) is cleaved specifically during apoptosis in mammalian cells (Kaufmann et al., Cancer Res, 53:3976-3985 1993) and is an excellent substrate in vitro for several ICE homologues (Tewari et al., Cell 81:801-809, 1995; Nicholson et al., Nature 376:3743 1995; Gu et al., J. Biol. Chem. 270:18715-18718, 1995; Fernandes-Alnemri et al., Cancer Res. 55:2737-2742 1995-, Fernandes-Alnemri et al., ibid.; Lippke et al., J. Biol. Chem. 271:1825-1828 1996). In the human promyelocytic leukemia cell line HL60 (Collins et al, Nature 270:347-349 1977), PARP is degraded in response to incubation with etoposide, which leads to cell death by apoptosis (Kaufmann et al., ibid). Protease inhibitors which block the activity of ICE homologues prevent not only apoptosis, but PARP degradation as well (Schlegel et al., ibid.).
Due to the inadequacies in many of the known methods for the detection of cell apoptosis, there continues to be a need for new, selective methods of detection. The present invention is directed to this, as well as other, important ends.